Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What subcellular structures and signaling pathways are important for rapid cell migration? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce normal cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, and live-cell phase-contrast or confocal time-lapse microscopy. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. We previously established that the topography of the extracellular matrix (ECM) plays a vital role in regulating cytoskeletal organization, cell morphology, and cell migration by demonstrating that one-dimensional (1D) micropatterned lines mimic the functions of the fibrillar ECM structures found in three-dimensional cell-derived matrix. We extended these studies to establish the mechanism by which fibrillar topography evokes rapid, efficient cell migration in fibroblasts and how this mode of migration differs from migration studied previously using regular two-dimensional (2D) tissue culture substrates. We found that two key processes of mesenchymal cell migration, protrusion of the leading edge and adhesions formed within the lamella, are enhanced during 1D migration and are controlled indirectly by cellular contractility. These studies also established that 1D adhesions and 3D adhesions to cell-derived matrix fibrils are more stably associated with the matrix, consistent with prolonged cell adhesiveness. We are continuing to explore the role of ECM topography and physical properties such as stiffness in regulating fibroblast adhesion, migration, and mechanotransduction. These ongoing studies are comparing fibroblast responses to 3D collagen hydrogels of differing architecture in order to characterize the dynamics of cell adhesions to collagen fibrils of differing thickness and compliance, as well as determining the mode of cell migration in different types of collagen matrix that correspond to different types of extracellular matrix in vivo. We are also evaluating the regulatory and functional crosstalk between actomyosin contractility and microtubule post-translational modification in cell adhesion, migration, matrix assembly, and organ branching in development. Our studies are identifying a homeostatic balance between actomyosin-mediated contraction and the level of microtubule acetylation. This balance affects fibronectin matrix assembly, cell migration, and the effectiveness of embryonic organ branching. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of the precise manner in which cells move and interact with their matrix environment will also facilitate tissue engineering studies.